Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p < 0.05, **p < 0.01 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( B ) H&E staining (a and d) and immunohistochemsitry (b,c and e–g) of proBDNF in the foot skin at 3 h post-formalin injection. proBDNF is expressed in the epidermis, basal layer and subcutaneous layers in the foot skin (b,c); Higher magnification (box in b) showing proBDNF is also mildly expressed in the nerve fibers in the control plantar (c); Responding to peripheral inflammation by 5% formalin intra-plantar injection, intensive proBDNF immunoreactivity is observed and mainly localized in the inflammatory cells (f, black arrows) and nerve fiber-like structures (g). Scale bars: 50 μm, 3 replicates, n = 3 per group. ( C ) a, Representative Western blot of proBDNF and mBDNF; b–d, Semi-quantitative analyses of mBDNF, proBDNF and their ratio in the inflamed tissue after Complete Freund Adjuvant (CFA, 10 μL) intra-plantar injection into Kunming mice (*p < 0.05, **p < 0.01versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( D ) Histological staining (a) and proBDNF immunohistochemistry (b,c) in the plantar at 1 day post-CFA injection; c, higher magnification of box in b showing that proBDNF is highly expressed in the inflammatory cells. Scale bar, 100 μm, 3 replicates, n = 3 per group. Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Western Blot, Injection, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P < 0.05, **p < 0.01 versus vehicle, two-way ANOVA with Bonferroni’s Multiple Comparison post hoc test, n = 8 per group); ( B ) 1 st - and 2 nd - phase (* P < 0.05, **p < 0.01 versus vehicle, student’s t test, n = 8 per group). ( C ) Attenuation of abdominal writhing both in male and female Kunming mice by poly-Ab-proBDNF pretreatment (**p < 0.01 versus vehicle, student’s t test, n = 12 per group).

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Injection

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P < 0.05, **p < 0.01 versus baseline, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 10–12 per group). ( B ) Ectopic overexpression of proBDNF by intra-plantar injection of Ad-proBDNF or Ad-EGFP reduces PWT dramatically in Kunming mice. (a) Representative proBDNF Western blot (upper panel) and its semi-quantitative analysis (lower panel, *P < 0.05, ***p < 0.001 versus control, ### p < 0.001 versus indicated groups, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test, n = 4 per group); (b) Representative fluorescent images after delivery of Ad-EGFP . Scale bar, 50 μm, 3 replicates, n = 3 per group; (c) PWT at 7 days post-injection of Ad-proBDNF or Ad-EGFP control (***p < 0.001 versus Ad-EGFP , student’s t test, n = 7 per group). ( C ) Co-injection of proBDNF (0.1 μg), but not mBDNF (0.1 μg) restored the biphasic nociceptive response after low-concentration of formalin (0.5%) intra-plantar injection (*p < 0.05versus vehicle, two-way ANOVA followed by Bonferroni’s Multiple Comparison post hoc test, n = 10–12 per group). ( D ) Exogenous proBDNF (1 μg) intra-plantar injection induces ERK activation in the ipsilateral spinal cord dorsal horn at 3 h post-injection, Scale bar, 50 μm, n = 3 per group, 3 replicates. ( E ) Spinal p-ERK expression at 3 h after proBDNF (1 μg) intra-plantar injection. (a) Representative Western blot and (b,c) their semi-quantitative analyses of p-ERK, (*p < 0.05, **p < 0.01 versus proBDNF-R, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Injection, Over Expression, Western Blot, Concentration Assay, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p < 0.001 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( B ) p75NTR immunohistochemistry in the local tissue at 3 h after 5% formalin intra-plantar injection. Image in lower panel is the higher magnification of box in the upper panel. Scale bar, 50 μm, 3 replicates, n = 3 per group. ( C ) H&E staining of local tissue 3 h after exogenous proBDNF (1 μg) injection, Scale bars, 100 μm, 3 replicates, n = 3 per group. ( D ) Representative Western blot of local cytokines IL-1β, IL-6 and TNF-α expression and their semi-quantitative analysis at 3 h and 24 h after 1 μg exogenous proBDNF intra-plantar injection (*p < 0.05, **p < 0.01, ***p < 0.001 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 4 per group). ( E ) Time course of IL-1β, IL-6 and TNF-α gene expression 3h after 0.25 μg and 1 μg proBDNF injection (*p < 0.05, **p < 0.01, ***p < 0.001 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Western Blot, Expressing, Injection, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P < 0.001 versus control, # p < 0.05 versus indicated group, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test). Neurospheres treated with proBDNF (100 ng/mL) showed dormancy without any neuronal migration and neurospheres had no morphological changes. Neurospheres treated with 2B11 (100 ng/ml) showed strong migration capability comparing with other groups. Neurospheres treated with 2B11 and proBDNF (100 ng/ml) showed similar ability of migration with sheep anti-proBDNF antibody treatment group.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: Human Molecular Genetics

Article Title: The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aβ

doi: 10.1093/hmg/ddv130

Figure Lengend Snippet: The pro-domain of BDNF and Aβ 1–42 exhibits synergistic toxicity in SH-SY5Y human neuroblastoma cell cultures. ( A ) Addition of 25, 100 and 1000 n m oligomeric Aβ 1–42 to the culture medium of SH-SY5Y human neuroblastoma cells for 48–60 h resulted in significant toxicity when compared with vehicle. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05. ( B ) Cells were treated with 200 ng/ml recombinant pro-domain of BDNF for 48–60 h. Met66 BDNF pro-domain alone did not have an effect on cell viability in control cells. ( C ) Cells were treated with Met66 pro-domain of BDNF (200 ng/ml) in combination with 25 n m Aβ 1–42 for 48–60 h. In the presence of Aβ 1–42 cell death was increased upon addition of Met66 BDNF pro-domain ( n = 9) performed. Each bar represents mean ± SEM ( n = 9). Statistical comparisons were made by paired t test. ** P < 0.01. ( D ) Twenty-six paired experiments indicated that cell death was higher when a particular preparation of Aβ 1–42 (25 n m , n = 9 preparations of Aβ 1–42 ) was supplemented with Met66 BDNF pro-domain ( y -axis) than for that preparation of Aβ 1–42 alone ( x -axis, dashed line indicates the null hypothesis of no effect of the Met66 BDNF pro-domain). ( E ) Similar results were seen when non-differentiated and ( F ) differentiated cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 1 μ m Aβ 1–42 for 48 h. ( G ) Cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 25 n m Aβ 1–42 oligomers for 48–60 h before SDS–PAGE and western blotting with antibodies against p75 NTR and sortilin. β-Actin expression was used as a loading control. Western blots indicated that levels of p75 NTR but not sortilin were higher following Aβ 1–42 oligomer treatment in cultures of human neuroblastoma cells. Representative blots are shown ( n = 3). ( H ) The p75 NTR and ( I ) sortilin intensities from the western blots were normalized against the level of β-actin. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05.

Article Snippet: Twenty four hours after plating, cells were washed and maintained in 0.5% (v/v) FBS-containing medium for 18 h. After that cells were replaced with 0.2% (v/v) FBS-containing medium and then incubated further 48–72 h in the presence of Aβ 1–42 oligomers with or without recombinant human wild type (Val66) or variant (Met66) BDNF pro-domain (Alomone labs).

Techniques: Recombinant, SDS Page, Western Blot, Expressing